This invention relates to the field of molecular biology. In particular, this invention relates to horse endothelin-B receptor genes and gene products and methods of analysis of the genes and gene products.
Coat color genetics plays an important role in mapping studies in mouse, rat and horse. Barsh, G. S. (1996) TIG Vol. 12, No. 8 299-305; Bowling, A. T., (1987) Journal of Heredity. 78: 248-250; and Marklund et al., (1996) Mammalian Genome 7: 895-899. Because coat color forms are desirable and selectable qualities in domestic species, variants are often preserved and recorded by a registry. The American Paint Horse Association registers Paint horses, as well as those thoroughbreds or offspring of Quarter Horse parents that are born with a white spotting pattern.
There are two distinct coat color patterns recognized by the American Paint Horse Association: tobiano and overo. Tobiano, inherited as an autosomal dominant trait, has been mapped to a linkage group containing Albumin and Vitamin D binding protein that is conserved in mouse and humans (Bowling, 1987). The overo inheritance pattern is not clearly defined and the map position has not been identified, perhaps because there are genetically different patterns registered as xe2x80x9coveroxe2x80x9d. One particular pattern registered as overo is characterized by a variable pattern of jagged white markings predominantly localized to the lateral and ventral parts of the body and is referred to as xe2x80x9cframexe2x80x9d overo. Other patterns registered as overo exist where all four legs and the head are white but there is little white on the lateral aspect of the horse. Clear distinction between patterns is not always possible from phenotype so these patterns have been registered as a single category (overo). Frame overos are the specific subset of overos that most frequently appear to produce foals that exhibit Lethal White Foal Syndrome. McCabe et al., American Jour. Med. Gen. 36:336-340 (1990); Trommershausen-Smith, Theriogenology 8(5):303-311 (1977); Vonderfecht et al., Amer. Jour. Med. Gen. (1983).
Lethal White Foal Syndrome is a disease in horses characterized by an all white coat and intestinal tract abnormalities that result in colic and related symptoms within 12 hours of birth. McCabe et al., (1990); Trommershausen-Smith, (1977); Vonderfecht et al., 1983). White foals with the disease die soon after birth. There is no known treatment or cure for this syndrome. Bowling, A.T. (1994) Journal of Heredity. 85 (3):222-224; McCabe, L., et. al. (1990) Amer. Jour. Med. Gen. 36:336-340; Vonderfecht, S. L., et al. (1983) Vet. Patho. 20: 65-70.
Diseases similar to Lethal White Foal Syndrome have been identified and genetically characterized in humans (Hirschsprungs Disease) Chakravarti, A. Human Molecular Genetics 5:3:303-307 (1996) and mice (piebald lethal). Metallinos, D. L., et al. (1994) Genetics. 136: 217-223. These diseases have been demonstrated to be caused by mutations in the endothelin-B receptor gene of humans and mice, respectively. The horse endothelin-B receptor gene has not been identified nor has it been shown to be related to Lethal White Foal Syndrome.
There is thus a need to identify and clone the Lethal White Foal gene in horses in order to identify carriers of the gene. In addition, there is a need to clone the horse endothelin-B receptor gene and to determine if mutations in the gene cause Lethal White Foal Syndrome. There is also a need to develop a molecular genetic test for Lethal White Foal Syndrome so that horses that have the potential to produce lethal white foals can be identified. There is a further need to place the gene for Lethal White Foal Syndrome on the horse genetic map so that it can be used as an anchor locus in the continued development of a horse syntenic map.
In order to meet these needs, the present invention is directed to the identification, isolation, cloning, sequencing, purification and mapping of the horse (Equine) wild type and mutant endothelin-B receptor (EDNRB) gene and cDNA.
This invention is also directed to molecular biological probes including DNA, RNA, cDNA, peptide, protein and antibody probes for horse EDNRB genes and gene products and mutant genes and gene products.
This invention is also directed to a PCR assay for the detection of EDNRB gene mutations in horses.
This invention is also directed to methods of in vivo and in vitro analysis of the expression of horse EDNRB wild type and mutant genes and gene products.
This invention is further directed to isolated oligonucleotides which include one or more nucleotide sequences and the corresponding complement nucleotide sequences selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24 and SEQ ID NO: 25.
This invention is further directed to a polymerase chain reaction (PCR) kit which includes oligonucleotides such as one or more of: SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15. The kit of the invention may further include reaction buffers and PCR DNA polymerase.
This invention is further directed to a method for determining the expression of the wild type and mutant horse endothelin-B receptor gene in a biological sample by nucleic acid hybridization. In the method of the invention, a nucleic acid probe including one or more of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24 and SEQ ID NO: 25 is utilized in hybridization reactions.
This invention is further directed to a method for identifying a horse wild type and mutant endothelin-B receptor gene by amplifying a portion of the endothelin-B receptor gene from a horse biological sample by using primers such as SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15 in amplification reactions wherein the amplification results in the generation of 174, 105 and 90 nucleotide-long polynucleotides. The method of the invention further includes the step of detecting the presence of amplification products.
The invention is further directed to an isolated nucleotide sequence comprising the sequence shown in SEQ ID NO: 20. The invention is further directed to the nucleotide sequence shown in SEQ ID NO: 20 wherein the sequence is linked to a heterologous nucleic acid. In the invention, the heterologous nucleic acid may be a promoter and/or a vector which is capable of functioning in mammalian, microbial, plant or avian cells.
The invention is further directed to an isolated nucleic acid encoding the polypeptide of SEQ ID NO: 21.
The invention is further directed to purified or isolated peptides comprising the amino acid sequence shown in SEQ ID NO: 21, SEQ ID NO: 17 and SEQ ID NO: 19. The invention is further directed to isolated and purified polyclonal and monoclonal antibodies produced from the peptide sequences of SEQ ID NO: 21, SEQ ID NO: 17 and SEQ ID NO: 19.